Abstract
Baicalein, showing higher bioavailability and stronger pharmacological activity, can be obtained via a β-glucuronidase (GUS)-catalyzed transformation of baicalein 7-O-β-D-glucuronide (baicalin). Recently, we have found that the fermentation broth of Lacticaseibacillus rhamnosus HP-B1083 can efficiently convert baicalin to baicalein. In this study, the L. rhamnosus HP-B1083-derived enzyme involved in baicalin biotransformation was identified and characterized. First, the LruidA gene, encoding the responsible enzyme, was cloned and sequenced. Sequence analysis revealed that the deduced enzyme (designated as LrUidA) belonged to the glycosyl hydrolase family 2. The recombinant LrUidA was expressed and purified for characterization. LrUidA had a molecular weight of 70 kDa, with an optimal temperature of 50 °C and pH 4.5. Although LrUidA was susceptible to temperature, it possessed a relative pH stability. Its Michaelis-Menten constant, maximum reaction velocity and catalytic constant values were 9.710 mM, 13.08 mM/min/mg, and 14.95 s-1, respectively. Site-directed mutagenesis experiment results demonstrated that the enzyme reaction uses side chains of E509 and E415 to hydrolyze the glycosidic bond of baicalin and involves three negatively charged residues, E450, D451, and D452, respectively. Surprisingly, biotransformation was performed under optimized reaction conditions by incubating the purified enzyme with 0.1 % baicalin for 4 h, resulting in a considerable conversion ratio of 99 %. Altogether, our findings provide insights into the properties of L. rhamnosus HP-B1083-derived enzyme and expand our understanding regarding using GUS for the industrial production of baicalein.